Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase

Structural Maintenance of Chromosomes (SMC) complexes are important for many aspects of the chromosomal organization. Unlike cohesin and condensin, the SMC5/6 complex contains a variant RING domain carried by its Nse1 subunit. RING domains are characteristic for ubiquitin ligases, and human NSE1 has been shown to possess ubiquitin-ligase activity in vitro. However, other studies were unable to show such activity. Here, we confirm Nse1 ubiquitin-ligase activity using purified Schizosaccharomyces pombe proteins. We demonstrate that the Nse1 ligase activity is stimulated by Nse3 and Nse4. We show that Nse1 specifically utilizes Ubc13/Mms2 E2 enzyme and interacts directly with ubiquitin. We identify the Nse1 mutation (R188E) that specifically disrupts its E3 activity and demonstrate that the Nse1-dependent ubiquitination is particularly important under replication stress. Moreover, we determine Nse4 (lysine K181) as the first known SMC5/6-associated Nse1 substrate. Interestingly, abolition of Nse4 modification at K181 leads to suppression of DNA-damage sensitivity of other SMC5/6 mutants. Altogether, this study brings new evidence for Nse1 ubiquitin ligase activity, significantly advancing our understanding of this enigmatic SMC5/6 function.

Nuclear envelope reshaping around the vacuole determines the morphology of the ribosomal DNA

The ribosomal DNA array (rDNA) of Saccharomyces cerevisiae has served as a model to address chromosome organization. In cells arrested before anaphase (mid-M), the rDNA acquires a highly structured chromosomal organization referred to as the rDNA loop, whose length can double the cell diameter. Previous works established that complexes such as condensin and cohesin are essential to attain this structure. Here, we report that the rDNA loop adopts distinct morphologies that arise as spatial adaptations to changes in the nuclear morphology triggered during the mid-M arrests. Interestingly, the formation of the rDNA loop results in the appearance of a space under the loop (SUL) which is devoid of any nuclear component yet colocalizes with the vacuole. We finally show that the formation and maintenance of the rDNA loop and the SUL require TORC1 and membrane synthesis. We propose that the rDNA-associated nuclear envelope (NE) reshapes into a loop to accommodate the vacuole, with the nucleus becoming bilobed.

CYTOLOGICAL VARIATION OF (AAG)7 REPEAT ON LETTUCE CHROMOSOMES BY FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

Cytological studies using fluorescence in situ hybridization (FISH) technique provides phylogenetical information in closely related taxa and have been widely applied for karyotyping and studying chromosomal organization and evolution in plant species. In the present study, FISH using a microsatellite sequence of (AAG)7 as the probe was performed in order to discriminate the chromosomes in four Lactuca species, i.e., L. sativa, L. serriola, L. saligna, and L. virosa. The experiment was carried out in April to September 2018 at Laboratory of Genetic and Plant Breeding of Breeding of Graduate School of Horticulture, Chiba University, Japan. Different distribution patterns of (AAG)n signals were shown on the chromosomes in the four Lactuca species studied, In L. sativa and L. serriola, FISH with (AAG)7 sequences revealed dispersed distribution patterns with one pair of bright signals, respectively. While in L. saligna and L. virosa, distinct signals with different intensities were observed in two pairs of chromosomes of L. saligna and five pairs of chromosomes of L. virosa. In conclusion, the AAG repeat signals could be used as cytogenetic landmarks for chromosome identification in Lactuca species.

Physical modeling of the heritability and maintenance of epigenetic modifications

We develop a predictive theoretical model of the physical mechanisms that govern the heritability and maintenance of epigenetic modifications. This model focuses on a particular modification, methylation of lysine-9 of histone H3 (H3K9), which is one of the most representative and critical epigenetic marks that affects chromatin organization and gene expression. Our model combines the effect of segregation and compaction on chromosomal organization with the effect of the interaction between proteins that compact the chromatin (heterochromatin protein 1) and the methyltransferases that affect methyl spreading. Our chromatin model demonstrates that a block of H3K9 methylations in the epigenetic sequence determines the compaction state at any particular location in the chromatin. Using our predictive model for chromatin compaction, we develop a methylation model to address the reestablishment of the methylation sequence following DNA replication. Our model reliably maintains methylation over generations, thereby establishing the robustness of the epigenetic code.